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Pathological and immunohistochemical studies for diagnosis of infectious agents associated with reproductive tract in small ruminants Sran Ravneet Kaur1, Mahajan Vishal2,*, Leishangthem Geeta Devi1, Bal Mandeep Singh2, Filia Gursimran2, Banga Harmanjit Singh1 1Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India 2Animal Disease Research Centre, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India *Address for Correspondence, Dr Vishal Mahajan, Animal Disease Research Centre, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India, E-mail: mahajanv17@gmail.com
Online Published on 28 July, 2022. Abstract The present study was conducted to decipher important and specific infectious agents affecting the reproductive tract of small ruminants viz. Brucella abortus, Chlamydophila abortus and Neospora caninum in Punjab using gross, histopathological and immunohistochemical techniques. A total of 11 aborted fetuses, 15 placental tissues and 26 female reproductive tracts (uterus) were collected from multiple locations. Immunohistochemical (IHC) examination revealed positive immunoreactivity for Brucella abortus and Chlamydophila abortus in placenta and uterus sections of small ruminants. For Brucella, 6/15 placental tissues, 3/16 uterus and 1/6 fetal tissues were positive by IHC. For Chlamydia, 3/15 placental tissues, 3/16 uterus were positive. No positive immunoreactivity was seen for Neospora in the present study. Histopathogical changes were co-related with IHC findings. It was found that microscopical changes ranged from chronic to mixed inflammatory cell infiltration in tissues infected with Brucella, whereas in chlamydial infections, tissues showed mixed cell infiltration to neutrophilic infiltration. Altogether, it is suggested that the immunohistochemical technique supplements the conventional methods of diagnosis of above mentioned diseases. Top Keywords Histopathology, Immunohistochemistry, Reproductive diseases, Small ruminants. Top | Introduction Sheep and goat are very important livestock species having versatile production profile due to their ability to convert forages, crops and household residues into meat, fiber, skin, wool, manure and milk. Small ruminants serve as the predominant source of supplementary income and livelihood for small and marginal farmers representing mostly the poorer sections of society in Punjab. Any pathology of the female reproductive tract especially uterus contribute to poor reproductive performance, hence reduced enterprise profitability1. Various infections of the pregnant uterus usually cause placentitis2 which is usually associated with abortion, stillbirths or weak offspring depending on the severity and stage of infection. About 90% of the diagnosed causes of abortion are infectious in nature and most important abortifacient agents are Brucella abortus, Chlamydophila abortus and Neospora caninum. |
An important ancillary technique to the many histochemical stains is immunohistochemistry (IHC) as it enables for a more standardized and more sensitive diagnostic work-up of samples3. It also demonstrates the distribution of organism in tissues. The present study presents the pathological and immunohistochemical findings in small ruminants from Punjab state, India, naturally infected with abortifacient agents viz. Brucella abortus, Chlamydophila abortus and Neospora caninum. |
Top Materials and Methods For pathological studies, tissue samples viz. involving female reproductive tract of small ruminants (uterus, n=26), placenta (n=15) from animals with history of abortion and aborted foetuses (n=11) were collected in 10% neutral buffered formalin from slaughter houses, Necropsy facility of GADVASU and farm outbreaks or clinical cases. |
For IHC, female reproductive tract (uterus, n= 16), placenta (n= 15) and aborted fetuses (n= 6) were processed. Serial sections were mounted on Poly-L-lysine coated microscopic slides in duplicate and kept in hot air oven for 1 hour at 60°C. The slides were deparaffinized, rehydrated and subjected to antigen retrieval by incubation in citrate buffer (pH 6.0) for 3 minutes at 100°C followed by 7 minutes at 70°C in Enzyme retriever. After cooling, slides were washed in PBS. The activity of non-specific proteins in tissue sections was blocked by incubating with 2.5% normal horse serum for 30 min (Vector, ImmPRESS™ reagent kit, Burlingame, CA, USA). All sections were then incubated overnight at 4°C with the primary antibody (Invitrogen Monoclonal antibody (mAb) for B. abortus, diluted 1:20; Invitrogen mAb for Chlamydia, diluted 1:25 and VMRD, Pullman mAb for Neospora caninum, diluted 1:25). Next day after PBS washing, endogenous peroxidase was blocked by immersing the sections in 3% hydrogen peroxide prepared in absolute methanol for 15 min. Tissue sections were again washed with PBS followed by 30-minute incubation of slides with a drop of horseradish peroxidase–conjugated polyclonal secondary antibody (Vector, ImmPRESS™reagent peroxidase kit, Burlingame, USA). After another PBS wash 3, 3-diaminobenzidine (DAB) solution was used as chromogen, and Gill’s hematoxylin was used for counterstaining. Finally, the slides were passed through alcohol and xylene and mounted with DPX. The negative control sections were incubated with horse serum instead of the primary antibody. The slides were examined and photographed using light microscope. |
Top Results Brucellosis The aborted fetuses collected in this study were present at various stages of gestation (Fig. 1). Grossly, fetus showed accumulation of sero-sanguinous fluid in thoracic cavity and necrotic foci in liver. The abomasal contents were yellowish, thick and cloudy with flecks of fibrin. Histopathologically, bronchopneumonia with predominantly lympho-mononuclear cell infiltration and occasional neutrophils was observed mainly within bronchiolar and alveoli lumen. Fetal liver showed multiple foci of lympho-mononuclear cell infiltration and fatty degenerative changes in hepatocytes. Placenta samples revealed haemorrhagic and necrotic cotyledons. Microscopically, edema of chorionic stroma, marked congestion and haemorrhages were observed. Trophoblast cells of placenta showed vacuolar degeneration, chronic hyperplastic changes and exfoliation. Chronic placentitis was characterised by infiltration of lympho-mononuclear cells and plasma cells (Fig. 2). Mixed inflammatory cell infiltration was also seen in few placental tissues comprising a population of plasma cells, lympho-mononuclear cells and neutrophils (Fig. 3). Uterus samples collected in this study showed thickened endometrium which was due to chronic endometritis confirmed microscopically. Sub-epithelial layer of endometrium and lamina propria was infiltrated diffusely by lympho-mononuclear cells (Fig. 4). Sloughing of epithelium was seen with haemorrhages underneath. Endometrial glands showed cystic degeneration (Fig. 5). Diffuse mixed cell infiltration was also seen in few uterine sections with congested blood vessels, edema and fibrosis around glands (Fig. 6). Myxomatous degeneration with rarefied endometrial glands surrounded by edema, proliferation of fibroblasts and infiltration of neutrophils predominantly was observed in uterus sample (Fig. 7). Immunohistochemical reaction for B. abortus was seen in one fetal lung, intra-cytoplasmically within alveolar macrophages of lungs present around and inside lumen of alveoli (Fig. 8). Six of the 15 placenta samples showed brown granular immunoreactivity in cytoplasm of trophoblast cells of placenta (Fig. 9, 10). Bacterial antigen was also detected in three uterus samples, inside cytoplasm of inflammatory cells present around endometrial glands in lamina propria (Fig. 11). Chlamydiosis The aborted fetuses from the late gestation showed coagulative necrosis of hepatocytes surrounded by scant mononuclear cell infiltrates. Placenta showed necrotic cotyledons appearing darker red grossly (Fig. 12). Histopathologic changes in placenta were suggestive of necro-suppurative placentitis with mineralisation and congestion (Fig. 13). Vacuolar degeneration of trophoblasts was seen with edema in chorion. Few placenta sections showed non-specific inflammatory response with mixed cell infiltration comprising of polymorphonuclear cells, plasma cells and mononuclear cells. Vasculitis was seen in placental sections with thickened tunica media due to infiltration of neutrophils and few mononuclear cells. Bacterial colonies were also seen. Uterus showed purulent exudate on gross examination. Microscopically, purulent endometritis was seen with marked infiltration by neutrophils in sub epithelial layer and around endometrial glands in lamina propria (Fig. 14). Other uterus tissues showed mixed cell infiltration with edema, congestion and fibrosis around endometrial glands. Positive immuno-reactivity of Chlamydophila was seen in three placenta sections as brown, granular immunoreaction in cytoplasm of trophoblast cells and also extracellularly (Fig. 15, 16). Three uterus samples tested positive by IHC with reaction seen in cytoplasm of inflammatory cells present in lamina propria around endometrial glands and also sub epithelially in endometrium (Fig. 17). Neosporosis Fetuses collected in mid gestation showed congested cerebellar hemispheres. Microscopical lesions in brain consisted of nonsuppurative encephalitis and marked congestion (Fig. 18). Perivascular cuffing was observed (Fig. 19). Satellitosis and neuronophagia along with spongiosis of grey matter was present. IHC detection for Neospora tissue cysts or tachyzoites in fetal or placental tissues failed to produce any results. Top Discussion Diagnostic accuracy is considerably improved when IHC technique is employed along with histological examination. Brucella antigens can be easily identified in aborted fetus or placentas by using IHC techniques4 and it is considered safer technique keeping in mind the use of formalin fixed tissues, than the culture techniques. In this study, both histopathological and IHC techniques were conducted on collected tissue samples. For Brucella, histopathological lesions observed in aborted fetuses in present study were similar to earlier studies4,5 who reported interstitial pneumonia or bronchopneumonia and periportal mononuclear infiltration in liver. IHC detected immunoreactivity in inflammatory cells of lungs. Greatest overload of bacteria is seen in macrophages of lungs of fetus because of the aspiration of amniotic fluid which contains the bacteria5. In the present study, placenta samples showed chronic inflammatory changes and hyperplasia. Edema was also seen along with mixed cell infiltration. IHC detected antigen in trophoblast cells. Brucella multiply within the cytoplasm and rough endoplasmic reticulum (RER) of trophoblasts thus causing its hypertrophy6. Chronic endometritis to diffuse mixed cell infiltration was seen in uterine sections with sloughing of epithelium, congestion, edema and fibrosis. Similar histopathological changes in uterus were reported by McCollum et al7. IHC detected antigen in cytoplasm of inflammatory cells present in endometrium and around glands. These results were similar to earlier investigations 1,7 who demonstrated similar immunoreactivity in macrophages inside the lumen of glands in endometrium. |
Necrotic placental cotyledons were observed in Chlamydia positive samples. Necro-suppurative placentitis with mineralisation, congestion and edema were observed in this study. Other changes like vasculitis and mixed cell inflammatory changes were also observed. Similar changes were reported by various authors. Livingstone et al8 noticed necrotic cotyledons appearing darker red with intercotyledonary area affected by edema, haemorrhagic foci and a diffuse infiltration of mixed inflammatory cells. Di Paolo et al9 reported thickened placenta with fibrinopurulent exudates in inter-cotyledonary spaces grossly and intracytoplasmic, basophilic inclusions in trophoblasts along with multifocal mineralization, microscopically. Hazlett et al10 and Giannitti et al11 reported suppurative and necrotizing placentitis with vasculitis, congestion and intracytoplasmic basophilic bacteria in trophoblastic cells of placenta because of phagocytic nature of trophoblasts. Positive immunoreactivity was seen in cytoplasm of trophoblast cells of placenta. Chlamydia was seen intra-cytoplasmically by IHC in trophoblast cells and elementary bodies were seen as small dots extracellularly9. Uterus samples in this study were suggestive of mixed cell inflammation and purulent endometritis. Antigen was demonstrated in inflammatory cells present in endometrium. Papp and Shewen12 observed lymphocytic and plasma cell infiltration in subepithelial layer of uterus from ewes infected chronically with C. psittaci and demonstrated Chlamydia antigen by IHC in subepithelial cells of uterus. |
For Neospora, pathological studies showed non suppurative encephalitis with foci of gliosis, congestion and perivascular cuffing in fetal tissue but tachyzoite or tissue cyst stages were not detected by H&E and IHC. Changes like non-suppurative encephalitis, edema of the Virchow-Robins spaces, infiltration of the media with mononuclear cells gliosis, necrosis, perivascular cuffing, haemorrhages have been reported in Neospora positive fetal brains13. Protozoal cyst or tachyzoites have been detected using Neospora specific IHC by Dubey and Lindsay14. Nakagaki et al15 reported severe lymphoplasmacytic perivascular cuffing and focal gliosis in CNS but N. caninum cysts were not seen by both histopathology and IHC. Chronic or subclinical infections in adult animals lead to development of immunity which interferes with cyst formation or lesions development16. |
In conclusion, the results of the present study revealed that placental cotyledons showed positive immunoreactivity for Brucella and Chlamydophila in maximum number of cases and thus placenta should preferably be submitted for diagnosis of abortion in small ruminants. Further, IHC combined with histopathology serves as a reliable diagnostic tool. |
Top Figures | Fig. 1.: Aborted fetuses at various stages of gestation
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| | Fig. 2.: Chronic placentitis with lympho-mononuclear cell infiltration along with marked congestion. H&E Bar, 100 μm
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| | Fig. 3.: Placenta showing mixed cell infiltration comprising a population of plasma cells, mono-nuclear cells and neutrophils. H&E Bar, 100 μm
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| | Fig. 4.: Chronic endometritis seen with diffuse infiltration of lympho-mononuclear cells in endometrium. H&E Bar, 100 μm
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| | Fig. 5.: Uterus showing cystic degeneration of glands. H&E Bar, 100 μm.
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| | Fig. 6.: Diffuse infiltration of mixed type inflammatory cells in uterus along with congestion, fibrosis and edema around glands. H&E Bar, 100 μm
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| | Fig. 7.: Rarefied endometrial glands due to myxomatous degeneration. Edema, fibroblasts and neutrophils seen around glands. H&E Bar, 100 μm
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| | Fig. 8.: Positive immunoreactivity with anti-Brucella-monoclonal antibody in lung. IHC Bar, 20 μm
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| | Fig. 9.: Positive immunoreactivity with anti-Brucella-monoclonal antibody in trophoblast layer of placenta. IHC Bar, 100 μm
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| | Fig. 10.: Positive immunoreactivity with anti-Brucella-monoclonal antibody in trophoblast layer of placenta. IHC Bar, 20 μm
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| | Fig. 11.: Positive immunoreactivity with anti-Brucella-monoclonal antibody in cytoplasm of inflammatory cell present around endometrial glands in lamina propria. IHC Bar, 20 μm.
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| | Fig. 12.: Necrotic placental cotyledons appearing darker red in color
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| | Fig. 13.: Necro-suppurative placentitis with neutrophilic infiltration and congestion. H&E Bar, 50 μm
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| | Fig. 14.: Uterus showing purulent endometritis with neutrophils infiltrating in sub epithelial layer and around endometrial glands along with loss of epithelium. H&E Bar, 50 μm
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| | Fig. 15.: Positive immunoreactivity with anti-Chlamyd-ia-monoclonal antibody in trophoblast layer of placenta. IHC Bar, 200 μm.
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| | Fig. 16.: Positive immunoreactivity with anti-Chlamydia-monoclonal antibody in trophoblast layer of placenta. IHC Bar, 200 μm
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| | Fig. 17.: Positive immunoreactivity with anti-Chlamydia-monoclonal antibody in cytoplasm of inflammatory cells present in lamina propria of uterus. IHC Bar, 20 μm
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| | Fig. 18.: Brain showing multi-focal gliosis along with congested blood vessels and spongiosis. H&E Bar, 50 μm
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| | Fig. 19.: Perivascular cufffing in brain. H&E Bar, 50 μm.
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